烧伤大鼠离体肺泡巨噬细胞CD14的表达及分泌IL-6的调控研究

目的探讨严重烧伤对大鼠肺泡巨噬细胞(AM)CD14膜蛋白(CD14)和mRNA基因表达变化的影响,以及抗CD14抗体对AM产生白细胞介素-6(IL-6)的调控作用。方法20%Ⅲ°烧伤大鼠检测早期外周血内毒素(LPS)浓度。体外分离培养AM,分成烧伤血清组、血清抗体组、内毒素组、内毒素抗体组。RT-PCR方法观察膜表面CD14 mRNA表达、免疫组织化学方法观察蛋白含量及ELISA方法观察分泌IL-6的变化。烧伤血清刺激体外培养的正常大鼠AM,观察培养上清中IL-6浓度变化及CD14抗体的抑制作用。结果烧伤后外周血LPS各时相点LPS浓度均明显高于对照组,与此相对应,烧伤组大鼠AM各时相点CD14 mRNA表达、蛋白含量均明显增高,AM培养上清中IL-6浓度亦显著增高(P<0.01)。以烧伤血清与AM培养1 h后,烧伤组培养上清中IL-6浓度增加值明显高于对照组(P<0.01),而在CD14抗体存在时,IL-6浓度增加值明显小于烧伤组(P<0.01)。结论严重烧伤后外周血LPS浓度增加,AM膜表面CD14受体亦显著增加,使LPS对免疫系统的激活作用显著增大,AM分泌IL-6明显增加。提示严重烧伤后可以通过调节CD14的作用而减少炎性介质的合成和分泌。     

人肠上皮细胞内毒素低反应性及其机制探讨

为探讨人正常肠上皮细胞对内毒素（LPS）刺激低反应性的机制，用ELISA法检测LPS刺激肠上皮细胞18h后IL－8的分泌水平，用RT－PCR及RPA法检测肠上皮细胞LPS跨膜信号转导相关受体TLR4，TLR2，CD14和MD2 mRNA的表达及LPS刺激对其表达的影响，结果表明：LPS刺激不能引起肠上皮细胞IL－8的分泌，而IL－1β刺激却能引起其分泌；人正常肠上皮细胞TLR4，TLR2，CD14和MD2mRNA的表达均较弱，LPS刺激后，TLR4，CD145 CD14和MD2 mRNA表达进一步降低，而TLR2的表达无显著变化，说明肠上皮细胞对LPS的低反应性与细胞表达LPS相关受体低表面有一定的内在联系，从而进一步证实TLR4，CD14，MD2在介导LPS跨膜信号转导中起重要作用。     

AP—2在内毒素刺激大鼠肺泡巨噬细胞中的动态变化及其在TNF—α表达中的调控作用

为观察内毒素刺激对大鼠肺泡巨噬细胞（AM）中转录因子AP－1活性的影响，探讨AP－1在LPS刺激大鼠肺泡巨噬细胞表达TNF－α的基因调控作用，应用凝胶迁移阻滞技术（EMSA）检测LPS（E coli 026:B6)刺激大鼠AM核蛋白中AP－1的DNA结合活性的动态变化，在LPS刺激前12h，应用转基因技术将AP－1的硫代寡核苷酸圈套（S－ODN decoy)转入大鼠AM，应用ELISA法观察AP－1硫代寡核苷酸圈套对LPS刺激大鼠AM表达TNF－α的影响，结果发现，应用100ng/ml LPS刺激大鼠AM 0．5h，AP－1即迅速出现活化并达到峰值，在1h活性略有下降，3h活性又逐渐回升，5h,8h又迅速回落，AP－1的活化状态至少可以持续8h，且与LPS刺激呈明显的量效关系；AP－1的S－ODN圈套能明显抑制但不能完全阻断LPS诱导的TNF－α表达，说明LPS刺激可使大鼠AM内AP－1活化，其在LPS介导的炎症反应中可能发挥重要作用，AP－1在LPS诱导的大鼠AM表达TNF－α中可能起重要的调控作用，但TNF－α表达可能还与其他核转录因子有关。     

脂多糖结合蛋白对内毒素急性肺损伤大鼠肺巨噬细胞TLR4信号通路的影响

为研究脂多糖结合蛋白（LBP）对内毒素（LPS）急性肺损伤大鼠TLR4肺巨噬细胞信号通路的影响，探讨LBP对LPS炎症反应增敏作用的部分机制，运用RT-PCR和ELISA方法分别检测LBP对LPS刺激后大鼠肺巨噬细胞IL-1β、IL-18mRNA表达与TNF-α分泌量的作用，同时用RT-PCR与Western blot检测其TLR4mRNA与蛋白的表达水平。结果显示，LBP显著增加LPS刺激后大鼠肺巨噬细胞TNF-α的分泌和IL-1β、IL-18mRNA的表达，同时也增加TLR4的mRNA与蛋白表达水平，而LBP多抗能阻断上述作用。说明LBP可增强由TLR4介导的LPS信号跨膜转导功能，这可能是LBP对LPS起增敏作用的重要分子机制之一。     

核因子—κB在内毒素刺激大鼠肺泡巨噬细胞中的动态变化及其在肿瘤坏死因子—α表达中的作用

目的 观察内毒素(LPS)刺激对大鼠肺泡巨噬细胞(alveolar macrophages，AM)中核因子—κB(NF—κB)活性的影响，探讨NF—κB在LPS刺激大鼠AM表达肿瘤坏死因子—α(刀NF—α)中的作用。方法体外观察LPS刺激大鼠AM中NF—κB活性的动态变化，应用圈套策略观察NF—κB在LPS刺激大鼠AM表达刀NF—α中的作用。结果LPS刺激可使大鼠AM内NF—κB明显活化，并与LPS剂量正相关；抗体超迁移率实验结果显示p50、p65参与了NF—κB的活化；NF—κB的圈套硫代寡核苷酸(S—ODN)能明显抑制LPS刺激大鼠AM表达TNF—α，但不能完全阻断LPS诱导的TNF—α表达。结论 NF—κB(p50／p65)在LPS介导的炎症反应中发挥重要作用，LPS刺激大鼠AM表达TNF—α受NF—κB调控，但其他核转录因子可能也在TNF—α的表达中发挥重要作用。     

AP-1在内毒素刺激大鼠肺泡巨噬细胞中的动态变化及其在TNF-α表达中的调控作用

为观察内毒素刺激对大鼠肺泡巨噬细胞(AM)中核转录因子AP-1活性的影响,探讨AP-1在LPS刺激大鼠肺泡巨噬细胞表达TNF-α中的基因调控作用,应用凝胶迁移阻滞技术(EMSA)检测LPS(E coli 026:B6)刺激大鼠AM核蛋白中AP-1的DNA结合活性的动态变化;在LPS刺激前12h,应用转基因技术将AP-1的硫代寡核苷酸圈套(S-ODN decoy)转入大鼠AM,应用ELISA法观察AP-1硫代寡核苷酸圈套对LPS刺激大鼠AM表达TNF-α的影响.结果发现,应用100ng/ml LPS刺激大鼠AM 0.5h,AP-1即迅速出现活化并达到峰值,在1h活性略有下降,3h活性又逐渐回升,5h、8h又迅速回落,AP-1的活化状态至少可以持续8h,且与LPS刺激呈明显的量效关系;AP-1的S-ODN圈套能明显抑制但不能完全阻断LPS诱导的TNF-α表达.说明LPS刺激可使大鼠AM内AP-1活化,其在LPS介导的炎症反应中可能发挥重要作用;AP-1在LPS诱导的大鼠AM表达TNF-α中可能起重要的调控作用,但TNF-α表达可能还与其他核转录因子有关.     

核因子-κB在内毒素刺激大鼠肺泡巨噬细胞中的动态变化及其在肿瘤坏死因子-α表达中的作用

目的　观察内毒素 (LPS)刺激对大鼠肺泡巨噬细胞 (alveolarmacrophages,AM)中核因子 -κB(NF -κB)活性的影响 ,探讨NF -κB在LPS刺激大鼠AM表达肿瘤坏死因子 -α(TNF -α)中的作用。　方法　体外观察LPS刺激大鼠AM中NF -κB活性的动态变化 ,应用圈套策略观察NF-κB在LPS刺激大鼠AM表达TNF -α中的作用。　结果　LPS刺激可使大鼠AM内NF -κB明显活化 ,并与LPS剂量正相关 ;抗体超迁移率实验结果显示p5 0、p6 5参与了NF -κB的活化 ;NF -κB的圈套硫代寡核苷酸 (S -ODN)能明显抑制LPS刺激大鼠AM表达TNF -α ,但不能完全阻断LPS诱导的TNF -α表达。　结论　NF -κB(p5 0 p6 5 )在LPS介导的炎症反应中发挥重要作用 ,LPS刺激大鼠AM表达TNF -α受NF -κB调控 ,但其他核转录因子可能也在TNF -α的表达中发挥重要作用。     

杀菌性/通透性增加蛋白模拟肽对内毒素致血管内皮细胞损伤的保护作用

目的：通过杀菌性／通透性增加蛋白（BPI）模拟肽（BNEP）拮抗内毒素（LPS），观察对血管内皮细胞分泌白细胞介素－6（IL－6），肿瘤坏死因子－α（INF－α）以及表达细胞间黏附分子－1（ICAM－1）的影响，探讨BNEP对LPS致血管内皮细胞损伤的保护作用。方法：采用体外培养的原代人脐静脉内皮细胞（HUVEC）为模型，分别观察单纯应用LPS，BNEP以及不同剂量BNEP与LPS作用后各细胞因子的分泌表达，IL－6，TNF－α应用双抗夹心酶联免疫吸附法（ELISA）测定。ICAM－1采用生物素－抗生物素－过氧化物酶复合物（SABC）－cy3免疫荧光染色，激光共聚焦扫描显微镜下观察其表达情况，结果：LPS 10ng/ml可使IL－6分泌量显著增加；BNEP 60ug/ml即可显著降低10ng/ml LPS诱导的IL－6分泌，随BNEP浓度增加IL－6分泌量呈降低趋势，达240ug/ml时，IL－6分泌量与单纯应用BNEP组比较，差异无显著性意义（P＞0．05），BNEP 120ug/ml与LPS10ng/ml作用后，可使TNF－α分泌量降低94％，BNEP 240ug/ml可完全阻断TNF－α的分泌。激光共聚焦扫描显微镜观察结果显示，BNEP与LPS作用后，ICAM－1在细胞膜及细胞浆的表达明显减弱。结论：BNEP在阻断LPS对细胞的激活和损伤，减少炎症介质和细胞因子的过度释放中具有明显的保护作用。     

烧伤血清诱导RAW264.7细胞分泌TNF-α的规律

目的：探讨烧伤血清诱导小鼠单核巨噬细胞系RAW264．7细胞分泌TNF-α的规律及其在脓毒症中的作用。方法：烧伤血清的剂量效应组分别用含有0．5,1．0,1．5,2．0,2．5ml烧伤血清的培养基与小鼠单核巨噬细胞系RAW264．7共培养6h,用双抗体夹心ELISA法测其上清中的TNF-α含量。采用流式细胞技术观察细胞的凋亡。烧伤血清的时间效应组以含有2．5ml烧伤血清的培养基,对照组以不含烧伤血清的培养基,另外以含有2．5ml烧伤血清和100mg／L LPS的混合组的培养基分别培养RAW264．7细胞0,2,4,6,8,10,12,24h,同上留取离心后的培养液上清,用双抗体夹心ELISA法测其上清中的TNF-α含量。结果：将烧伤血清加入到小鼠单核巨噬细胞RAW264．7培养液中能明显刺激TNF-α的分泌。在0．5ml～2．5ml范围内,烧伤血清诱导RAW264．7细胞的TNF-α分泌增加,呈显著的剂量依赖性关系,2．5ml烧伤血清能明显诱导RAW264．7细胞的凋亡;烧伤血清组和烧伤血清与LPS的混合组RAW264．7细胞的TNF-α分泌在0～24h间呈双相性规律。结论：烧伤血清能诱导小鼠单核巨噬细胞系RAW264．7的TNF-α分泌与凋亡,初步证实烧伤血清在脓毒症的发生、发展中发挥重要作用。     

烧伤血清对肠上皮细胞屏障功能损伤的实验研究

目的 系统观察烧伤血清对肠上皮细胞屏障功能的影响。方法 制作体外大鼠肠上皮细胞IEC－6 烧伤血清刺激模型，实验分为对照组和烧伤血清组，采用生化检测、图像分析、细胞ELISA、放射免疫等技术，动态观察肠上皮细胞活性、细胞Ca^2 浓度，细胞通透性以有细胞骨架的变化。结果 肠上皮细胞经烧伤血清作用后，细胞活性即开始降低，而乳酸脱氢酶升高，胞浆Ca62 浓度增高2．43倍，单层细胞通透性显著增加，细胞骨架成分肌动蛋白（F－actin）、角蛋白（keratin）、微管蛋白（β－tubulin）表达减少或状态改变。结论 烧伤血清刺激后肠上皮细胞活性降低，通透性增加，细胞骨架表达减少，从而导致肠黏膜细胞屏障功能受损。     

Knee injury and Osteoarthritis Outcome Score (KOOS) - validation of a Swedish version

The Knee injury and Osteoarthritis Outcome Score (KOOS) is a self-administered instrument measuring outcome after knee injury at impairment, disability, and handicap level in five subscales. Reliability, validity, and responsiveness of a Swedish version was assessed in 142 patients who underwent arthroscopy because of injury to the menisci, anterior cruciate ligament, or cartilage of the knee. The clinimetric properties were found to be good and comparable to the American version of the KOOS. Comparison to the Short Form-36 and the Lysholm knee scoring scale revealed expected correlations and construct validity. Item by item, symptoms and functional limitations were compared between diagnostic groups. High responsiveness was found three months after arthroscopic partial meniscectomy for all subscales but Activities of Daily Living.     

Endovascular management of carotid-cavernous fistulas

Objective To investigate endovascular treatment of traumatic direct carotid-cavernous fistulas （CCF） and their complications such as pseudoaneurysms. Methods： Over a five-year period, 22 patients with traumatic direct CCFs were treated endovascularly in our institution. Thirteen patients were treated once with the result of CCF occluded, 8 twice and 1 three times. Treatment modalities included balloon occlusion of the CCF, sacrifice of the ipsilateral internal carotid artery with detachable balloon, coll embolization of the cavernous sinus and secondary pseudoaneurysms, and covered-stem management of the pseudoaneurysms. Results All the direct CCFs were successfully managed endovascularly. Four patients developed a pseudoaneurysm after the occlusion of the CCF with an incidence of pseudoaneurysm formation of 18.2% （4/22）. A total number of 8 patients experienced permanent occlusion of the ICA with a rate of ICA occlusion reaching 36.4% （8/22）. Followed up through telephone consultation from 6 months to 5 years, all did well with no recurrence of CCF symptoms and signs. Conclusion Traumatic direct CCFs can be successfully managed with endovascular means. The pseudoaneurysms secondary to the occlusion of the CCFs can be occluded with stent-assisted coiling and implantation of covered stents.     

The acutely limping child

Acute limping may be the result of multiple pathologies in children. The differential diagnosis varies based on the age of the child. Irrespective of age, the initial imaging work-up includes AP and frog leg radiographs of the pelvis and ultrasound; MRI may sometimes be helpful. In children less than 3 years, infections and trauma are most frequent. MRI is the imaging modality of choice when osteomyelitis is clinically suspected. Between the ages of 3 and 10 years, transient synovitis of the hip and Legg-Calvé-Perthes disease are main considerations but infection, inflammation and focal bony lesions are also considered. In children over 10 years, slipped capital femoral epiphysis also is considered.     

Do Balance Board Training Programs Reduce the Risk of Ankle Sprains in Athletes?

Introduction Ankle sprains are the most common musculo-skeletal injury that occurs in athletes,particularly in sports that require jumping and landing on one foot such as soccer,and basketball(1-4).These injuries often result in significant time loss from participation,long-term disability,and have a major impact on health care costs and resources(5-8).     

High Intensity Interval Training:New Insights

KEY POINTS ·High-intensity interval training(HIT)is characterized by repeated sessions of relatively brief，intermittent exercise.often performed with an“a11 out”effort or at an intensity close to that which elicits peak oxygen uptake(i.e.，≥90%of VO2 peak).     

Developmental validation of the PowerPlex(®) ESI 16 and PowerPlex(®) ESI 17 Systems: STR multiplexes for the new European standard

In response to the ENFSI and EDNAP groups’ call for new STR multiplexes for Europe, Promega^® developed a suite of four new DNA profiling kits. This paper describes the developmental validation study performed on the PowerPlex^® ESI 16 (European Standard Investigator 16) and the PowerPlex^® ESI 17 Systems. The PowerPlex^® ESI 16 System combines the 11 loci compatible with the UK National DNA Database^®, contained within the AmpFlSTR^® SGM Plus^® PCR Amplification Kit, with five additional loci: D2S441, D10S1248, D22S1045, D1S1656 and D12S391. The multiplex was designed to reduce the amplicon size of the loci found in the AmpFlSTR^® SGM Plus^® kit. This design facilitates increased robustness and amplification success for the loci used in the national DNA databases created in many countries, when analyzing degraded DNA samples. The PowerPlex^® ESI 17 System amplifies the same loci as the PowerPlex^® ESI 16 System, but with the addition of a primer pair for the SE33 locus. Tests were designed to address the developmental validation guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM), and those of the DNA Advisory Board (DAB). Samples processed include DNA mixtures, PCR reactions spiked with inhibitors, a sensitivity series, and 306 United Kingdom donor samples to determine concordance with data generated with the AmpFlSTR^® SGM Plus^® kit. Allele frequencies from 242 white Caucasian samples collected in the United Kingdom are also presented. The PowerPlex^® ESI 16 and ESI 17 Systems are robust and sensitive tools, suitable for the analysis of forensic DNA samples. Full profiles were routinely observed with 62.5 pg of a fully heterozygous single source DNA template. This high level of sensitivity was found to impact on mixture analyses, where 54–86% of unique minor contributor alleles were routinely observed in a 1:19 mixture ratio. Improved sensitivity combined with the robustness afforded by smaller amplicons has substantially improved the quantity of data obtained from degraded samples, and the improved chemistry confers exceptional tolerance to high levels of laboratory prepared inhibitors.